Module 3. Genomic data and annotations in public databases Exercises Custom sequence annotation

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1 Module 3. Genomic data and annotations in public databases Exercises Custom sequence annotation Objectives Upon completion of this exercise, you will be able to use the annotation pipelines provided by the Influenza Research Database (IRD), Virus Pathogen Resource (ViPR) and PATRIC to annotate your own sequences. I. Annotate Influenza Virus segment sequences 1. Influenza virus segment sequence annotation You can annotate your influenza sequences using IRD s unique sequence curation/annotation pipeline, which will determine the influenza type, segment number, subtype (if appropriate), translated amino acid sequence(s), and any potential sequencing artifacts for each input sequence. a. Go to > Analyze & Visualize > Annotate Nucleotide Sequences. b. Input the sample H5 sequence (Influenza_H5_AM fasta) in the workshop package you downloaded: click Choose File > locate the file. Then click Validate Sequence(s). SEARCH DATA ANALYZE & VISUALIZE WORKBENCH SUBMIT DATA HELP Home Annotate Nucleotide Sequences Annotate Nucleotide Sequences This tool is an interactive version of the IRD Team's influenza annotation pipeline. You can submit any number of nucleotide sequences in FASTA format for validation and annotation. The pipeline will align the sequences against a consensus sequence profile to identify possible sequencing errors, determine the influenza type, segment number, and for segments 4 and 6 of type A, the subtype, and translate the nucleotide sequence. You will be provided with a report containing these results and a list of possible sequencing errors or, if critical errors are encountered, a description of the errors. This pipeline was developed by Dr. Catherine Macken, currently at the University of Auckland, and is maintained by Dr. Macken and the IRD team. Either paste or upload segments in FASTA format below and click the Validate Sequence(s) button. Note: An asterisk (*) = required field Submitter E mail Notice when annotation Completed? Upload Your Segment Data * Enter your sequence in FASTA or Upload your sequence in FASTA File Influenza_H fasta Cancel Validate Sequence(s) c. After the annotation process is finished, a Sequence Annotation Result page will be loaded. Here you will see flu type, segment number, subtype (if you provided HA or NA sequences), and translated protein sequence. d. Note that IRD aligns the query sequence with profile sequences and provides warning messages if insertions, deletions or mismatches were found. e. Click Download Amino Acid FASTA and save the file to your computer for later exercises. Sequence Annotation Result YOUR TICKET NUMBER: Select/Deselect All Annotation Summary Report Detailed Annotation Report Download Amino Acid FASTA Click on + to view detailed annotation report. 1 sequences submitted 1 sequences pass auto curation without having any warning message(s) 0 sequences pass auto curation with warning message(s) 0 sequences fail auto curation with error message(s) Annotation Details: >AM Influenza A virus A/Anas acuta/slovenia/470/06 4 H5N1 Northern Pintail Flu Type: A Segment number: 4 Subtype: H5 Sequence Length: 1779 Submitted Sequence: AGCAAAAGCAGGGGTTCAATCTGTCAAAATGGAGAAAATAGTGCTTCTTC [full] Translated Protein Protein Name: HA Hemagglutinin Sequences: CDS Location: MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILE [full] 1

2. Highly pathogenic H5 virus clade classification IRD has a Highly Pathogenic H5N1 Clade Classification Tool, which can classify the clade of the HA gene of highly pathogenic H5 viruses.

2 2. Highly pathogenic H5 virus clade classification IRD has a Highly Pathogenic H5N1 Clade Classification Tool, which can classify the clade of the HA gene of highly pathogenic H5 viruses. The IRD algorithm has been verified as highly accurate (>99%) for 3/11/2016 Influenza Research Database - H5N1 High Pathogenic Clade Classification sequences of at least 300 nucleotides of HA1. a. Go to Analyze & Visualize > H5 Clade Classification. b. In the Input Sequences section, choose Analyze my custom sequences only > Choose File > locate the sample H5 sequence (Influenza_H5_AM fasta) in the workshop package you downloaded > Run. Loading Influenza AboutResearch Us Community Database... Announcements Links Resources Support Sign Out yun.zhang@jcvi.org SEARCH DATA ANALYZE & VISUALIZE WORKBENCH SUBMIT DATA HELP Home H5N1 Clade Classification Highly Pathogenic H5N1 Clade Classification Tool The IRD team has implemented an algorithm for classifying the clade of the hemagglutinin gene of influenza A(H5) viruses. It uses phylogenetic analysis to classify HA (H5) sequences (from both highly pathogenic Goose/Guangdong like viruses and from non pathogenic Eurasian and American lineage viruses) according to the WHO classification scheme. This algorithm was developed by IRD team member Catherine Macken, of the University of Auckland. It has been verified as highly accurate (> 98%) for sequences of at least 300 nucleotides of HA1. See SOP for more details. Sequences from other serotypes of HA, or other segments will yield unpredictable and likely incorrect results. If unsure of your sequence's segment or serotype, we suggest you use the IRD Sequence Annotation Tool found on the Analyze and Visualize menu by clicking the Annotate Nucleotide Sequences link. ANALYSIS NAME INPUT SEQUENCES Analyze my custom sequences only. Upload a file containing my sequences in FASTA format. File Path: Exercise ann e (1).fasta Paste sequences in FASTA format. Analyze my custom sequences and associated metadata with IRD sequences. Clear c. 5/22/2017 Inﬂuenza Research Database - H5N1 High Pathogenic Clade Classiﬁcation Run Release Date: Mar 11, 2016 After the annotation process is finished, a Clade Classification Report page will be loaded. Here you can: Loading Influenza Database... i. View the clade assigned toresearch your input sequence. ii. Download the tree file by clicking Download in Unified Tree. iii. Visualize the classification tree including your query sequence by clicking View Tree. This project is funded by the National Institute of Allergy and Infectious Diseases (NIH / DHHS) under Contract No. HHSN C and is a collaboration between Northrop Grumman Health IT, J. Craig Venter Institute, and Vecna Technologies. About Us Community Announcements Links Resources Support Sign Out yun.zhang@jcvi.org SEARCH DATA ANALYZE & VISUALIZE WORKBENCH SUBMIT DATA HELP Home H5N1 Clade Classification Results H5N1 Clade Classification Report Save Analysis Download Raw Result Sequence Identifier AM Influenza A virus A/Anas acuta/slovenia/470/06 4 H5N1 Northern Pintail iv. Clade Assignment Unified Tree Download Tree Viewer View Tree In the tree viewer, find your query sequence by typing AM in the Search box. Sequences containing this string will be highlighted in green. Is it placed within the bounds of a single clade? Release Date: May 22, 2017 This system is provided for authorized users only. Anyone using this system expressly consents to monitoring while using the system. Improper use of this system may be referred to law enforcement officials. This project is funded by the National Institute of Allergy and Infectious Diseases (NIH / DHHS) under Contract No. HHSN C and is a collaboration between Northrop Grumman Health IT, J. Craig Venter Institute, and Vecna Technologies. 1/1 2

3. Identify Sequence Features in custom sequences Influenza sequences in IRD are annotated with 3,543 experimentally characterized regions termed Sequence Features (SF), as well as the predicted

This annotation tool allows users to annotate their custom sequences with the locations of Sequence Features and to determine if their sequences carry particular Phenotypic Variant Types (PVT) in

3 3. Identify Sequence Features in custom sequences Influenza sequences in IRD are annotated with 3,543 experimentally characterized regions termed Sequence Features (SF), as well as the predicted phenotypic effects of selected sequence variations. This annotation tool allows users to annotate their custom sequences with the locations of Sequence Features and to determine if their sequences carry particular Phenotypic Variant Types (PVT) in which particular sequence substitutions are predicted to give phenotypic effects, such as increased virulence or antiviral drug resistance. a. Go to Analyze & Visualize > Identify Sequence Features in Segments. b. You can either paste your sequence to the sequence box or upload your sequence. For this exercise, click Choose File > locate the sample H5 sequence (Influenza_H5_AM fasta) in the workshop package you downloaded > Sequence Feature Type: Functional > click Run. SEARCH DATA ANALYZE & VISUALIZE WORKBENCH SUBMIT DATA HELP Home Identify Sequence Features in Segments Identify Sequence Features in Segments This component allows users to determine if their sequences contain experimentally characterized regions termed 'Sequence Features'(SF) defined for all influenza virus proteins in IRD according to this SOP. This tool also allows users to determine if their sequences carry particular Phenotypic Variant Types (PVT) in which particular sequence substitutions give rise to phenotypic consequences, such as increased virulence or antiviral drug resistance. Either paste or upload segments in FASTA format below and click the 'Annotate Sequence(s)' button. The following influenza proteins are currently supported by SFVT data. Warning: on average a given flu protein will have 130 Epitope, 20 Structural, 20 Functional and 10 Sequence Alteration Sequence Features. Submitter SEQUENCE FEATURE TYPE E mail Notice when annotation Completed? Upload Your Segment Data * Enter your sequence in FASTA or Upload your sequence in FASTA File Influenza_H fasta Cancel Annotate Sequence(s) c. After the annotation process is finished, a Sequence Annotation Result page will be loaded. Here you can download the spreadsheet report by clicking Download Sequence Feature Annotations. d. Open up the downloaded spreadsheet report to see the annotation result. e. Look for Sequence Features related to species adaptation or tissue tropism. Does this sequence carry any Phenotypic Variant Type (PVTs) associated with either of the phenotypes? 3

4 4. HA subtype numbering conversion In order to facilitate hemagglutinin (HA) protein sequence comparison across different HA subtypes, IRD has implemented an HA Subtype Numbering Conversion tool based on the cross-subtype HA numbering scheme proposed by Burke and Smith in PLoS One, 2014, 9:e This tool allows you to convert the coordinates of any HA protein sequences to coordinates of any other subtypes, in order to map functional domains or phenotype markers across subtypes. a. Go to Analyze & Visualize > HA Subtype Numbering Conversion. b. The tool landing page will be displayed. In the Input Sequences section, choose Analyze my custom sequences only > Choose File locate the H5 protein sequence you downloaded at step 1.e. c. Next, select H5mEA-nonGsGD, H7N3 and H7N7 in the Conversion Sequence Numbering Scheme list. d. Then click Convert Residue Numbering. SEARCH DATA ANALYZE & VISUALIZE WORKBENCH SUBMIT DATA HELP Home HA Subtype Numbering Conversion HA Subtype Numbering Conversion (beta) This tool allows users to renumber HA sequences according to a cross subtype numbering scheme proposed by Burke and Smith in Burke DF, Smith DJ A recommended numbering scheme for influenza A HA subtypes. PLoS One 9:e Burke and Smith's numbering scheme uses analysis of known HA structures to identify amino acids that are structurally and functionally equivalent across all HA subtypes, using a numbering system based on the mature HA sequence. Either paste or upload HA protein in FASTA formats and click the "Convert residue numbering" button to map your input sequence to the Burke and Smith numbering scheme. See the SOP for a detailed description of how the HA Subtype Numbering Conversion tool works. ANALYSIS NAME INPUT SEQUENCES Analyze my custom sequences only. Upload a file containing my sequences in FASTA format. CONVERSION SEQUENCE NUMBERING SCHEME Select one or more from the pick list to convert the existing position numbers to different subtype numbering scheme(s). File Path: Influenza_H5 otein.fasta Paste sequences in FASTA format. Analyze my custom sequences and associated metadata with IRD sequences. Clear Convert residue numbering e. The tool will first BLAST your HA sequence(s) against the Burke and Smith reference sequences and find the closest hit. Verify the BLAST result and click Continue. Sequence Annotation Result AM Influenza A virus A/Anas Sequence Name: acuta/slovenia/470/06 4 H5N1 Northern Pintail HA Hemagglutinin Virus Type: A H5c221 ( A/chicken/Egypt/0915 NLQP/2009 Closest Reference sequence: (HPAI) ) Segment: 4 Warning message: None Please verify the BLAST result; if the information is correct click 'Continue' Continue f. The HA Subtype Numbering Conversion Result page will be loaded, where you can: i. Visualize the alignment of your HA sequence with your selected subtype reference sequences by checking the checkbox for your query sequence and then clicking the Visualize Aligned Sequences button located above the alignment result. ii. In the Numbering Conversion Result table, view the corresponding coordinates of your H5 sequence in H5mEA-nonGsGD (low pathogenic), H7N3 (low pathogenic) and H7N7 (highly pathogenic) numbering schema. Note that your input sequence has RRKK insertion which is a signature motif of highly pathogenic viruses. iii. Download the Numbering Conversion Result table and the alignment (FASTA or text format) from the Download dropdown menu located above the alignment result. 4

II. Hepatitis C Virus Typing Hepatitis C Viruses (HCV) have diversified into seven major genotypes (1-7) over time. Each major genotype is further classified into genotype/subtypes, e.g., 1a, 1b, 1c, etc.

5 II. Hepatitis C Virus Typing Hepatitis C Viruses (HCV) have diversified into seven major genotypes (1-7) over time. Each major genotype is further classified into genotype/subtypes, e.g., 1a, 1b, 1c, etc. The genotype and subtype assignments are regularly updated by the International Committee on Taxonomy of Viruses (ICTV). As of June 2017, the number of confirmed genotypes/subtypes has increased to 86 (ICTV, 2017). In order to assist researchers in designating appropriate assignments for new HCV sequences using the ICTV genotype/subtype assignments, the ViPR team has developed an HCV Genotyping/Subtyping Tool. This tool predicts the most likely genotype/subtype for the query sequence by placing it into the HCV reference tree using pplacer and then analyzing the pplacer output using cladinator. a. Go to > Featured Viruses Hepatitis C Virus > Analyze & Visualize > Genotype-Recombination Detection. HCV Genotyping/Subtyping Tool (Beta) The HCV Genotype Determination tool classifies the genotype/subtype of HCV viruses, based on the HCV genotype/subtype assignments maintained by the International Committee on Taxonomy of Viruses (ICTV) SOP for HCV genotyping tool HCV Reference Alignment HCV Reference Tree. This tool infers the genotype/subtype for a query sequence from its position within a reference tree. The tool requires input sequences to be at least 400 bp. Short sequences will yield unpredictable and likely incorrect results. ANALYSIS NAME SOURCE OF SEQUENCES TO BE ANALYZED * Sequences can also be selected from search results or a working set in your workbench. Analyze sequences saved in working sets Analyze my custom sequences only. Upload a file containing my sequences in FASTA format. Paste sequences in FASTA format. Analyze my custom sequences and associated metadata with ViPR sequences. Clear Run 5

6 b. On the HCV Genotyping/Subtyping Tool page, you can view a description of the method (SOP for HCV typing tool), reference alignment and reference tree used in the typing tool. c. Input the sample HCV genome sequences (HCV_DQ164588_KU fasta) included in the workshop package. To do so, choose Analyze my custom sequences only > Choose File > locate the file > Run. d. After the analysis is finished, the HCV Genotyping/Subtyping Report page will be displayed. HCV Genotyping/Subtyping Report (Beta) (SOP) Save Analysis Your analysis contains 2 records Query Identifier Consensus Assignment Support Phylogenetic Tree KU a Input alignment (FASTA) Output tree (Newick) Subtype assignment (text) DQ a Input alignment (FASTA) Output tree (Newick) Subtype assignment (text) Report Here you can: View the ViPR predicted genotype/subtype. Both sequences are genotype 5 in the GenBank records. What is the ViPR consensus assignment for each sequence? Click View in the phylogenetic tree column to load the output tree used to type the sequence. Download the alignment (FASTA) used as the input to pplacer, the output tree (Newick) and the subtype assignment report (text) generated from cladinator. e. Click View in the phylogenetic tree column for each query sequence. f. Find the query sequence by typing # in the Search textbox. Assign a genotype/subtype for each query sequence based on the output tree, and then compare your own assignments with ViPR consensus assignments. g. Does the sequence length affect the genotyping/subtyping result? KU is a full length genome and has one placement in the 5a lineage with a probability score of 1.0. DQ is a short sequence of 543 bp and has two placements in the tree. The top hit falls into the 5a lineage and has a probability score of III. Mycobacterium tuberculosis genome annotation The Genome Annotation Service in PATRIC uses the RAST tool kit (RASTtk) to provide annotation of genomic features. a. Go to PATRIC ( At the top of any PATRIC page, find the Services tab and then click Genome Annotation. 6

For this exercise, input the sample TB sequence (TB_1421917.fna) included in the workshop package. To do so, cl

7 b. This will open up the Annotation Service landing page. You can upload contigs from an assembly job completed in PATRIC or from an external source. For this exercise, input the sample TB sequence (TB_ fna) included in the workshop package. To do so, click the folder icon for Contigs. c. This will open up the Choose or Upload a Workspace Object lightbox. Click the Upload to Current Folder icon above the table. d. On the Upload files to Workspace page, choose Upload type Contigs, click Select File and choose TB_ fna from your computer, and then click Start Upload. 7

8 e. After uploading is completed, you will be returned to the Genome Annotation page. Choose a Taxonomy Name Mycobacterium tuberculosis, label your job, choose an output folder and then click the Annotate button. Note that the default genetic code is 11, for Archaea and most Bacteria. The other option is genetic code 4, which should be used if Mycoplasma, Spiroplasma or Ureaplasma are the organisms used. f. A message of Annotation Job has been queued will appear to indicate that the job is now in the queue. g. You can check the status of the annotation job via the Jobs indicator at the bottom of the PATRIC page. h. Once the annotation is completed, the result will be saved into your Workspace. To view the result, in the navy navigation bar, click Workspaces > My Jobs. i. This will open the Job Status page. Click the desired job and then click View in the right control panel. 8

j. This will load the Job Result page. You will see a job description table at the top and a list of annotation files at the bottom of the page. Click View at the top of the page. k.

A recommended numbering scheme for influenza A HA subtypes. PLoS One. 2014, 9:e112302. 2. Noronha JM, et al.

9 j. This will load the Job Result page. You will see a job description table at the top and a list of annotation files at the bottom of the page. Click View at the top of the page. k. You will be taken to the Genome View page. Look through the data tabs to view the various types of annotations for the genome. References 1. Burke DF, Smith DJ. A recommended numbering scheme for influenza A HA subtypes. PLoS One. 2014, 9:e Noronha JM, et al. Influenza virus sequence feature variant type analysis: evidence of a role for NS1 in influenza virus host range restriction. J Virol. 2012, 86(10): International Committee on Taxonomy of Viruses (ICTV). HCV Classification Matsen FA, et al. pplacer: linear time maximumlikelihood and Bayesian phylogenetic placement of sequences onto a fixed reference tree. BMC Biomathematics. 2010, 11:538. PMID: Zmasek C. cladinator Brettin T, et al. (2015). RASTtk: a modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes. Scientific reports 5:

a. From the grey navigation bar, mouse over Analyze & Visualize and click Annotate Nucleotide Sequences.

a. From the grey navigation bar, mouse over Analyze & Visualize and click Annotate Nucleotide Sequences. Section D. Custom sequence annotation After this exercise you should be able to use the annotation pipelines provided by the Influenza Research Database (IRD) and Virus Pathogen Resource (ViPR) to annotate